A novel transporter involved in cobalt uptake

Cobalt is an essential component of a low molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1. We have found a new gene, nhlF, in the DNA region sandwiched between nhlBA encoding L-NHase and amdA encoding amidase, which are involved in the degradation of nitriles. The product of nhlF, NhlF, shows a significant sequence similarity with those of hoxN from Alcaligenes eutrophus, hupN from Bradyrhizobium japonicum, nixA from Helicobacter pylori, and ureH from Bacillus sp., which are considered to be involved in nickel uptake into these cells. Sequence and hydropathy plot analyses have shown that NhlF encodes a 352-amino acid (aa) protein with eight hydrophobic putative membrane-spanning domains. nhlF expression in R. rhodochrous ATCC 12674 and Escherichia coli JM109 confers uptake of (57)Co in their cells, but not of (63)Ni. The expression of both nhlF and nhlBA in R. rhodochrous ATCC 12674 exhibited higher NHase activity than nhlBA expression. These findings together with the inhibitory effect by uncouplers (CCCP and SF6847) for the cobalt uptake suggest that NhlF mediates the cobalt transport into the cell energy-dependently finally to provide L-NHase.

钴是红球菌（Rhodococcus rhodochrous）J1来源的低分子质量腈水合酶（low molecular-mass nitrile hydratase, L-NHase）的必需组分。我们在参与腈类降解的、编码L-NHase的nhlBA与编码酰胺酶的amdA之间的DNA区域中，发现了一个新基因nhlF。nhlF的编码产物NhlF与真养产碱杆菌（Alcaligenes eutrophus）的hoxN、日本慢生根瘤菌（Bradyrhizobium japonicum）的hupN、幽门螺杆菌（Helicobacter pylori）的nixA以及芽孢杆菌属（Bacillus sp.）的ureH的氨基酸序列具有显著相似性，而上述蛋白均被认为参与宿主细胞对镍的摄取。序列分析和亲水性图谱分析结果显示，NhlF是一条含352个氨基酸（amino acid, aa）的蛋白质，带有8个疏水的潜在跨膜结构域。将nhlF在红球菌ATCC 12674与大肠杆菌（Escherichia coli）JM109中表达后，宿主细胞可摄取(57)Co，但无法摄取(63)Ni。在红球菌ATCC 12674中同时表达nhlF与nhlBA时，其腈水合酶活性高于仅表达nhlBA的菌株。结合解偶联剂（CCCP与SF6847）对钴摄取的抑制效应，上述研究结果表明，NhlF以能量依赖的方式介导钴向细胞内的转运，最终为L-NHase提供所需的钴。